The hypothesis to be tested is that defects in regulatory regions of the insulin gene are responsible for at least some of the inherited susceptibility to diabetes. The focus will be on (1) identification of regulatory regions of insulin genes in experimental animals and cultured cells, and (2) a search for defects in human insulin genes by structural analysis. In vivo and in vitro experiments ae proposed to determine whether glucocorticoid hormones and various peptides (presumably acting through islet cAMP) regulate insulin biosynthesis through changes in proinsulin (PI) mRNA. The results will indicate whether glucocorticoid hormones (1) maintain fasted and fed levels of PI mRNA, (2) modulate the PI mRNA response to glucose or act independently, and (3) are the effects of glucose and glucocorticoids additive? Similar experiments will determine whethe cAMP modulates fasted and glucose stimuated dPI mRNA levels and whether glucocorticoid and cAMP effects are additive suggesting independent mechanisms. Molecular mechanisms will be evaluated by measuring transcription rates and mRNA stability. Specific regulatory regions will be defined by cloning hybrid insulin genes and creating transgenic mouse lines. Regulation of insulin gene expression in cultured human islets will also be studied. To define genes which control insulin biosynthesis, the diabetes mutation (db) in diabetes resistant (C57BL/6J) and prone (C57BL/KsJ) mice will be studied by genetic and molecular analyses to determine whether: (1) diabetes susceptibility in the KsJ mouse is controlled by a single or multiple genes and (2) is this gene(s) linked to the insulin gene? Potential defects in the insulin gene related to diabetes will be approached at the DNA level by blot hybridization analysis with selected oligonucleotide probes to search for transcription mutants, and by hybridization with single stranded probes and electrophoresis on denaturation gradients to detect single base changes. Using restrictions fragment polymorphism at the insulin gene locus, linkage with NIDDM will be evaluated in pedigrees. Because of the association of a particuar class of insulin allele with IDDM, linkage will be studied in families with multiple affected sibs. For selected IDDM and NIDDM families showing lack of linkage to the insulin gene, permanent lymphoblastoid cell lines will be established to test linkage to othe genetic loci. Two other studies will be initiated: (i) linkage analysis with the insulini receptor gene, and (ii) a multilocus analysis. In summary, the results of the experiments proposed should clearly define the role of the insulin gene in diabetes, and begin to define other genes which may be involved.